JP6589327B2 - Recombinant hydrogen-oxidizing bacteria and protein production method using the same - Google Patents

Description

  The present invention relates to a hydrogen-oxidizing bacterium capable of secreting and expressing the protein in a culture solution by introducing a target protein gene, and a method for producing the protein using the same.

  In recent years, protein production research using microorganisms and cells has been actively conducted. In particular, biopharmaceuticals that include proteins with large molecular weights and complex structures have higher specificity and affinity for target molecules than low-molecular-weight drugs, so they can handle diseases that cannot be handled by conventional drugs. It is predicted that future demand will increase as a possible medicine.

  When proteins used for biopharmaceuticals are industrially produced, they are mainly produced using genetic recombination techniques and cell culture techniques, and most of them use production systems using E. coli as a host. However, in the conventional production system using E. coli as the host, it is necessary to use expensive raw materials such as yeast extract, peptone, amino acids, vitamins, etc. in the culture medium, and the production of the target protein is low. There is a problem that the cost becomes high. Many of the production systems express the target protein in E. coli, and an operation for extracting the target protein from the E. coli body is required. In addition, there is a problem that the produced target protein is degraded by a proteolytic enzyme derived from the body of Escherichia coli and the purity is lowered, and the purification cost is increased due to refolding of the formed inclusion bodies.

  A production system using yeast or Bacillus subtilis as a host can secrete and express the target protein in the culture medium. However, it is necessary to use expensive raw materials such as yeast extract as in the system using E. coli as a host. There are also problems of degradation of the target protein by a host-derived proteolytic enzyme and an increase in purification cost due to a by-product derived from a nitrogen source contained in the culture solution.

  In addition to Escherichia coli, yeast, and Bacillus subtilis, a hydrogen-oxidizing bacterium Ralstonia eutropha is known as one of the hosts considered to be effective in substance production. Ralstonia eutropha has been reported to accumulate a large amount of PHB (polyhydroxybutyric acid) under conditions where supply of nitrogen, phosphorus, oxygen, etc. is restricted, and bacteria containing PHB in high-density culture under heterotrophic conditions It has been confirmed that the body yield reaches 281 g / L (Non-patent Document 1). In addition, it is known that Ralstonia eutropha can grow under autotrophic conditions in which carbon fixation is performed by energy generated by hydrogen oxidation, and high-density culture is possible even in culture using only inorganic salts and sugars. Up to now, substance production using Ralstonia eutropha as a host is not limited to PHB described above, but also 2-methylcitric acid (Non-patent Document 2), 2-HIBA (hydroxyisobutyric acid) (Non-patent Document 3), R- Examples such as 3-hydroxybutyric acid (Non-Patent Document 4) and cyanophycin (Non-Patent Document 5) have been reported. In addition, examples of mass production of protein-derived organophosphorus degrading enzymes from raw materials consisting only of inorganic salts and glucose have been reported (Non-patent Documents 6 and 7).

  Ralstonia eutropha is known to have a wide variety of types and numbers of membrane proteins and secreted proteins. From these, a transport protein effective for secretory expression of the target protein is screened and coexpressed with the target protein. Thus, an example in which a target protein is secreted and produced in a culture solution is known (Patent Document 1).

Japanese Patent Application No. 2014-048577

Ryu, HW. et al. Biotechnol. Bioeng. , (1997) 55, 28-32. Ewering, C.I. et al. , Metab. Eng. , (2006) 8, 587-602. Hoefel, T .; et al. , Appl. Microbiol. Biotechnol. , (2010) 88, 477-484 Shiraki, M .; et al. , J .; Biosci. Bioeng. , (2006) 102, 529-534. Diniz, SC. et al. Biotechnol. Bioeng. , (2006) 93, 698-717. Srinivasan, S .; et al. Biotechnol. Bioeng. , (2003) 55, 114-120 Barnard, GC. et al. , Protein. Expr. Purif. , (2004) 38, 264-271

  Bacteria belonging to the genus Ralstonia, represented by the hydrogen-oxidizing bacterium Ralstonia eutropha, are microorganisms that originally inhabit various environments such as hot springs and soils, and can grow under autotrophic conditions, such as functions related to the uptake of substances due to their environmental adaptability. Has been suggested to have developed. In fact, Ralstonia eutropha JMP134, which takes up and degrades aromatic compounds, Ralstonia metallidurans CH34, which has the ability to discharge heavy metals by heavy metal antiporters, Ralstonia solanacerum, which has a mechanism to release a large number of extracellular toxins, Ralstonia, which has an extracellular PHB depolymerase There are various species such as pickettii, and it is clear that proteins related to mass transport on the cell membrane are rich in diversity. In addition, as a result of genome information analysis, 12% of protein genes in the genome are involved in the transport of substances, which is more than 9% of E. coli and supports the diversity of membrane transport proteins in bacteria belonging to the genus Ralstonia eutropha It is the result.

  As described above, since membrane transport proteins are more abundant than existing recombinant microorganisms such as Escherichia coli, hydrogen oxidizing bacteria belonging to the genus Ralstonia are used as hosts for secretory expression of target proteins using membrane transport proteins as carriers. Promising. So far, as a transport protein gene effective for secretory expression of a target protein screened from the genome of a hydrogen oxidizing bacterium of the genus Ralstonia, the H16_A2820 gene derived from the Ralstonia eutropha H16 strain can be mentioned. In this case, the F16-binding protein can be secreted and produced in the culture medium by introducing a fusion gene of H16_A2820 gene and Fc-binding protein gene into hydrogen-oxidizing bacteria and co-expressing the transport protein and the target protein. Is possible. However, in previous reports, about 40% of the total amount of the target protein produced by the cells remains in the cells, and the amount of the target protein secreted into the culture solution is only over 60% (culture). Secretion rate into the liquid is less than 70%). For this reason, in the construction of an extraction / purification process only from the culture solution, there has been a problem that the target protein remaining in the cells becomes a loss.

  Therefore, an object of the present invention is to allow 70% or more of the target protein produced by the bacterial cells to be secreted and expressed outside the bacterial cells in a hydrogen-oxidizing bacterium into which the target protein gene has been introduced (secretion ratio into the culture solution of 70% or higher). Another object of the present invention is to provide a novel hydrogen-oxidizing bacterium and a method for producing a target protein using the hydrogen-oxidizing bacterium. In the present invention, by selecting and using a high-performance transport protein with a higher secretion ratio of the target protein, the microorganism with the secretion ratio of 70% or more, the production efficiency in the culture solution is improved, and the purity of the target protein is high. Expression is possible.

  As a result of intensive studies to solve the above problems, the present inventor introduced the H16_B0271 gene derived from Ralstonia eutropha H16 strain as a transport protein gene in the hydrogen-oxidizing bacterium into which the target protein gene was introduced. By co-expressing the target protein and the transport protein, it was found that 70% or more of the total amount of the target protein produced by the cells can be efficiently secreted and expressed in the culture solution (outside of hydrogen-oxidizing bacteria). It came to complete.

That is, this invention includes the following aspects.
(1) A hydrogen-oxidizing bacterium capable of expressing a protein obtained by introducing a target protein gene and a transport protein gene into a hydrogen-oxidizing bacterium, wherein the transport protein is a polypeptide comprising the sequence of SEQ ID NO: 10.
(2) The hydrogen-oxidizing bacterium according to (1), wherein the transport protein gene is a polynucleotide comprising the sequence set forth in SEQ ID NO: 8.
(3) The hydrogen-oxidizing bacterium according to (1) or (2), wherein the hydrogen-oxidizing bacterium is a genus Ralstonia.
(4) The hydrogen-oxidizing bacterium according to any one of (1) to (3), wherein the target protein is an Fc-binding protein.
(5) A method for secreting and expressing a target protein outside the hydrogen-oxidizing bacterium by culturing the hydrogen-oxidizing bacterium according to any one of (1) to (4) and co-expressing the target protein and a transport protein.
(6) A plasmid for secreting and expressing a protein of interest outside a hydrogen-oxidizing bacterium, comprising a polynucleotide encoding a polypeptide consisting of the sequence of SEQ ID NO: 10.

  Hereinafter, the present invention will be described in detail.

  In the present invention, the hydrogen-oxidizing bacterium used as a host is not particularly limited, but Ralstonia genus bacteria that can grow under autotrophic conditions and have developed functions related to substance uptake are preferable. Among them, Ralstonia eutropha is various. It can be said to be a particularly preferred hydrogen-oxidizing bacterium because it has environmental adaptability and has abundant functions and numbers of proteins involved in mass transport.

  The present invention is characterized in that when a target protein is expressed using a hydrogen-oxidizing bacterium as a host, a transport protein gene is also introduced into the hydrogen-oxidizing bacterium in addition to the target protein gene. As the transport protein, a protein having a function of releasing the target protein into the culture medium by co-expression with the target protein may be appropriately selected from secretory proteins or membrane proteins possessed by hydrogen-oxidizing bacteria. It is preferable to select a protein having a high molecular weight or a protein having a molecular weight of 40 kDa or less. In addition, when the full length of transport protein is 40 kDa or more, what is necessary is just to express as a transport protein of 40 kDa or less using only a highly hydrophilic area | region.

  An example of the transport protein is the N-terminal region (SEQ ID NO: 8) of H16_B0271 derived from chromosome 2. In the present invention, the H16_A2820 gene derived from the Ralstonia eutropha H16 strain, which is a transport protein gene having a performance with a maximum secretion ratio of more than 60%, is replaced with a transport protein that can secrete the target protein more efficiently, ie, the H16_B0271 gene. As a result, a secretory expression system for a target protein having a secretion ratio of 70% or more exceeding the existing method in a culture solution has been completed. Here, the secretion ratio refers to the ratio of the target protein secreted outside the cell (in the culture solution) out of the total amount of the target protein produced by the cell inside and outside the cell.

  The transport protein gene to be introduced into the hydrogen-oxidizing bacterium may be obtained by preparing the transport protein cDNA or the like using a DNA amplification method such as a PCR method after ligation by an appropriate method, or from the amino acid sequence of the transport protein. It may be obtained by artificial synthesis after conversion to a nucleotide sequence. When converting from an amino acid sequence to a nucleotide sequence, it is preferable to convert in consideration of the codon usage in the host to be transformed. Analysis of codon usage frequency can also be performed by using a public database (for example, Codon Usage Database on the website of Kazusa DNA Research Institute). An example of a transport protein gene is a polynucleotide comprising a sequence described in SEQ ID NO: 10, which is a polynucleotide encoding a transport protein composed of the amino acid sequence described in SEQ ID NO: 8 (N-terminal region of H16_B0271).

  When introducing the target protein gene and transport protein gene in the present invention, the target protein and transport protein are linked via an appropriate cleavage linker that is automatically cleaved by a host-derived protease present in the periplasm or outer membrane. Co-expressed as a fusion protein with In this way, the expressed fusion protein of the target protein and transport protein is transported to the periplasm and then cleaved by protease, so that it is automatically secreted into the culture medium without any other special treatment. This is because that. The cleavage linker used here is not particularly limited as long as it is cleaved by a host-derived protease, and examples thereof include prePhaZ1, which is a signal sequence of extracellular PHB-degrading enzyme of pelB of E. coli or Paucimonas lemignei. In addition, when introducing the target protein gene and the transport protein gene in the present invention, it is more preferable to introduce a phasin promoter upstream of the fusion protein gene of (target protein gene- (preferably cleavage linker) -transport protein gene). The phasin promoter is a promoter for producing phasin, which is a protein that covers the surface of PHB (polyhydroxybutyric acid) granules, and is known as a strong promoter in Ralstonia eutropha. In addition, since the fadin promoter is activated by depletion of nitrogen and phosphorus sources in the culture solution, no expensive inducer such as IPTG (isopropyl-β-thiogalactopyranoside) used in E. coli expression systems is added. In addition, high protein expression is possible.

  When producing the hydrogen-oxidizing bacterium of the present invention, the target protein gene and the transport protein gene are introduced into the hydrogen-oxidizing bacterium mainly by an expression method using a broad host vector and a genome recombination method using a suicide vector. is there. When Ralstonia eutropha is used as a hydrogen-oxidizing bacterium, pBBR1MCS2, pKT230, and pBHR1 can be exemplified as wide-area host vectors, and pJQ200mp18, pNHG1, and pLO1 can be exemplified as suicide vectors. In addition, gene transfer may be performed using a broad-area host vector and a suicide vector in combination.

  The hydrogen-oxidizing bacterium of the present invention can produce proteins from inexpensive raw materials such as inorganic salts as compared to the expression system of Escherichia coli. For example, organic nitrogen materials such as yeast extract and peptone that are commonly used in E. coli culture can be replaced with inorganic nitrogen materials such as ammonia, ammonium salts, nitrates, and nitrites. Moreover, as a carbon source, it can replace with inorganic nitrogen carbon raw materials, such as a carbon dioxide and carbonate, instead of gluconic acid or fructose. By using such inorganic salt raw material without contaminating proteins and expressing the target protein into the culture medium, a high-purity protein can be obtained in the culture supernatant only by the culturing process. Simple process can be simplified.

  The target protein that can be secreted and expressed using the hydrogen-oxidizing bacterium of the present invention is not particularly limited, and examples include human-derived proteins such as insulin, interferon, interleukin, antibody, erythropoietin, growth hormone, and their receptor proteins. can give. The target protein to be secreted and expressed using the hydrogen-oxidizing bacterium of the present invention may be a complete protein, a polypeptide composed only of a part important for the function of the target protein, or a further object. One or more amino acids constituting the protein may be deleted and / or inserted and / or substituted. Hereinafter, among the target proteins that can be secreted and expressed using the hydrogen-oxidizing bacteria of the present invention, Fc-binding proteins will be described in detail.

In the present specification, the Fc binding protein is an extracellular region of human FcγRI (specifically, in the case of natural human FcγRI, a region from the 16th glutamine to the 292nd histidine in the amino acid sequence shown in SEQ ID NO: 11). ) Or a protein constituting the extracellular region of human FcγRIIIa (specifically, in the case of natural human FcγRIIIa, the region from the 17th glycine to the 208th glutamine in the amino acid sequence shown in SEQ ID NO: 12) Say. However, it does not necessarily have to be the entire region of human FcγRI extracellular region or human FcγRIIIa extracellular region, and at least an antibody (immunoglobulin) among proteins (polypeptides) constituting human FcγRI extracellular region or human FcγRIIIa extracellular region The polypeptide of the area | region which can express the original function couple | bonded with Fc area | region of this should just be included. As an example of the Fc binding protein,
(I) a protein comprising an amino acid residue from at least the 16th glutamine to the 289th valine in the amino acid sequence of SEQ ID NO: 11,
(II) contains at least amino acid residues from the 16th glutamine to the 289th valine in the amino acid sequence of SEQ ID NO: 11, and one or more of the amino acid residues are substituted with other amino acid residues Inserted or deleted proteins,
(III) a protein comprising an amino acid residue from the 17th glycine to the 192nd glutamine in the amino acid sequence of SEQ ID NO: 12,
(IV) comprising at least the 17th glycine to the 192nd glutamine amino acid residue in the amino acid sequence of SEQ ID NO: 12, and at least one of the amino acid residues is substituted with another amino acid residue Inserted or deleted protein,
Is given.

Specific examples of the (II) include Fc-binding proteins disclosed in JP2011-206046A and JP2014-027916A. Specific examples of the above (IV) include the amino acid residues from the 17th to the 192nd in the amino acid sequence shown in SEQ ID NO: 12, and the following (1 ) To (40), and Fc-binding protein (Japanese Patent Application No. 2013-202245) in which at least one amino acid substitution has occurred.
(1) 18th methionine of SEQ ID NO: 12 is replaced with arginine (2) 27th valine of SEQ ID NO: 12 is replaced with glutamic acid (3) 29th phenylalanine of SEQ ID NO: 12 is replaced with leucine or serine (4) 30th leucine of SEQ ID NO: 12 is replaced with glutamine (5) 35th tyrosine of SEQ ID NO: 12 is replaced with any of aspartic acid, glycine, lysine, leucine, asparagine, proline, serine, threonine, histidine (6) The 46th lysine of SEQ ID NO: 12 is replaced with isoleucine or threonine (7) The 48th glutamine of SEQ ID NO: 12 is replaced with histidine or leucine (8) The 50th alanine of SEQ ID NO: 12 is replaced with histidine (9) The tyrosine at number 12 is aspartic acid or histidine (10) The 54th glutamic acid of SEQ ID NO: 12 is replaced with aspartic acid or glycine (11) The 56th asparagine of SEQ ID NO: 12 is replaced with threonine (12) The 59th glutamine of SEQ ID NO: 12 is replaced with arginine (13) 61st phenylalanine of SEQ ID NO: 12 is replaced with tyrosine (14) 64th glutamic acid of SEQ ID NO: 12 is replaced with aspartic acid (15) 65th serine of SEQ ID NO: 12 is replaced with arginine (16) The 71st alanine of No. 12 is substituted with aspartic acid (17) The 75th phenylalanine of SEQ ID No. 12 is substituted with leucine, serine or tyrosine (18) The 77th aspartic acid of SEQ ID No. 12 is substituted with asparagine (19) The 78th alanine of SEQ ID NO: 12 becomes serine (20) The 82nd aspartic acid of SEQ ID NO: 12 was replaced with glutamic acid or valine (21) The 90th glutamine of SEQ ID NO: 12 was replaced with arginine (22) The 92nd asparagine of SEQ ID NO: 12 was replaced with serine ( 23) 93th leucine of SEQ ID NO: 12 is replaced with arginine or methionine (24) 95th threonine of SEQ ID NO: 12 is replaced with alanine or serine (25) 110th leucine of SEQ ID NO: 12 is replaced with glutamine (26 ) The 115th arginine of SEQ ID NO: 12 was replaced with glutamine (27) The 116th tryptophan of SEQ ID NO: 12 was replaced with leucine (28) The 118th phenylalanine of SEQ ID NO: 12 was replaced with tyrosine (29) 119th lysine substituted with glutamic acid (30) sequence 120th glutamic acid of No. 12 is substituted with valine (31) 121st glutamic acid of SEQ ID No. 12 is substituted with aspartic acid or glycine (32) 151st phenylalanine of SEQ ID No. 12 is substituted with serine or tyrosine (33) The 155th serine of No. 12 is replaced with threonine (34) The 163rd threonine of SEQ ID NO: 12 is replaced with serine (35) The 167th serine of SEQ ID NO: 12 is replaced with glycine (36) The 169th position of SEQ ID NO: 12 (37) 171th phenylalanine of SEQ ID NO: 12 is replaced with tyrosine (38) 180th asparagine of SEQ ID NO: 12 is replaced with lysine, serine or isoleucine (39) of SEQ ID NO: 12 185th threonine is replaced by serine (40) 12 192 th glutamine substituted lysine

The present invention can achieve the following effects.
(1) The hydrogen-oxidizing bacterium of the present invention introduces the target protein gene and the H16_B0271 gene, which is a novel transport protein gene excellent in transport efficiency, and co-expresses the target protein and the transport protein so far. It is difficult to secrete and express in a culture solution of 70% or more of the total amount of the target protein produced by the cells.
(2) The method for producing a target protein using the hydrogen-oxidizing bacterium of the present invention can reduce the cost for producing the protein as compared with the conventional secretory expression method. In conventional methods for secreting and expressing a target protein into a culture solution using hydrogen-oxidizing bacteria as a host, less than 40% of the total amount of the target protein produced by the cell remains in the cell without being released into the culture. This has been a factor in reducing the production amount of the target protein in the culture solution and the recovery rate during purification. On the other hand, in the present invention, by using the H16_B0271 gene, which is a novel transport protein gene with more excellent transport efficiency, a microorganism production process with high productivity in the culture solution and high recovery efficiency during purification is enabled.

It is a figure which shows the result of having evaluated and compared the productivity of the Fc binding protein in a test tube culture, and the secretion to a culture solution by the hydrogen oxidation bacterium which introduce | transduced the Fc binding protein gene and the transport protein gene. Black is the amount expressed in the culture medium, and white is the amount expressed in the cells.

  Hereinafter, the present invention will be described in more detail with reference to examples using Fc-binding proteins as proteins and Ralstonia bacteria as hydrogen-oxidizing bacteria. However, the present invention is limited to the above examples. It is not a thing.

Example 1 Preparation of Expression Vector Plasmids (expression vectors) shown in the following (a) to (c) were prepared.
(A) Phasin promoter, (H16_A2935 gene-cleaved linker gene-Fc binding protein gene) fusion gene, and plasmid introduced terminator gene (a-1) Phadine promoter, (H16_A2935 gene-cleaved linker gene-Fc binding property A gene in which a gene (sequence number 1) linked in the order of a fusion gene of a protein gene and a terminator gene was inserted into a plasmid pUC57 was prepared by artificial gene synthesis (Gen Script, artificial gene synthesis contract service, pUC57 is Gen Script) Provided artificial gene insertion standard vector). In SEQ ID NO: 1, the 7th to 444th regions are cleaved by the phasin promoter (SEQ ID NO: 2), the 451th to 684th regions are the H16_A2935 gene (SEQ ID NO: 3), and the 691st to 798th regions are cleaved. In the linker gene (SEQ ID NO: 4), the region from 805 to 1641 corresponds to the Fc binding protein gene (SEQ ID NO: 5), and the region from 1648 to 1758 corresponds to the terminator gene (SEQ ID NO: 6). In addition, a promoter derived from Ralstonia eutropha H16 strain was used as the fasin promoter, PrephaZ1 which is a signal sequence of the extracellular PHB-degrading enzyme of Pacimonas lemignei was used as the cleavage linker, and the H16_A2935 gene, the cleavage linker gene and the Fc-binding protein gene were used as Ralstonia eutropha, respectively. The nucleotide sequence is optimized for codons in the H16 strain. Hereinafter, this plasmid is referred to as A2935-FcR / pUC57.

(A-2) Escherichia coli JM109 strain was transformed with the prepared A2935-FcR / pUC57. The obtained transformant was cultured, and plasmid A2935-FcR / pUC57 was extracted.
(A-3) Escherichia coli JM109 strain was transformed with pBBR1MCS2, which is a commercially available broad-area host vector, and pBBR1MCS2 was prepared from the resulting transformant culture with a plasmid purification kit.
(A-4) The obtained pBBR1MCS2 was digested with ApaI and XbaI, and after agarose electrophoresis, a DNA product of about 5.1 kbp was purified by a gel extraction kit.
(A-5) A2935-FcR / pUC57 prepared in (a-2) was digested with restriction enzymes with ApaI and XbaI, and after agarose electrophoresis, the obtained DNA fragment of about 1.8 kbp was purified with a gel extraction kit. .
(A-6) The DNA fragment obtained in (a-4) and the DNA fragment obtained in (a-5) were subjected to a ligation reaction (Ligation High, manufactured by Toyobo Co., Ltd.) at 16 ° C. to obtain Escherichia coli JM109 strain (Takara Bio Inc.) was transformed with the plasmid.
(A-7) An LB plate culture solution (bactotryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) containing 20 μg / mL kanamycin and 1% glucose (w / v) was added to the solution after transformation. L, bactoagarose 15 g / L), and the target clone was selected.
(A-8) A plurality of selected colonies were cultured, and plasmid extraction was performed to confirm cleavage patterns by various restriction enzymes.

  By the above method, A2935-FcR / pBBR1MCS2, which is a plasmid in which the ApaI / XbaI fragment of plasmid A2935-FcR / pUC57 was inserted into the ApaI / XbaI site of pBBR1MCS2, was obtained. A2935-FcR / pBBR1MCS2 is a plasmid in which a fazine promoter, a fusion gene of (H16_A2935 gene-cleavable linker gene-Fc binding protein gene) and a terminator gene are inserted in the positive direction with respect to LacZ of pBBR1MCS2. The transformant obtained by transforming E. coli JM109 with A2935-FcR / pBBR1MCS2 is hereinafter referred to as A2935-FcR / JM109.

(B) Phasin promoter, (H16_A2820 gene-cleavable linker gene-Fc binding protein gene) plasmid and terminator gene-introduced plasmid (b-1) Ralstonia eutropha Plasmid A2820 / pUC57 in which a polynucleotide (SEQ ID NO: 9) containing a nucleotide sequence obtained by codon conversion (region 7 to 330 of SEQ ID NO: 9) was inserted into plasmid pUC57 was prepared by artificial gene synthesis ( Gen Script artificial gene synthesis contract service).
(B-2) Escherichia coli JM109 strain was transformed with the prepared A2820 / pUC57, and plasmid A2820 / pUC57 was extracted from the culture of the obtained transformant.
(B-3) A2935-FcR / pBBR1MCS2 prepared in (a) was digested with restriction enzymes HindIII and SpeI, and after agarose electrophoresis, the obtained DNA fragment of about 6.6 kbp was purified by a gel extraction kit.
(B-4) A2820 / pUC57 prepared in (b-2) was digested with restriction enzymes HindIII and SpeI, and after agarose electrophoresis, the obtained DNA fragment of about 0.3 kbp was purified by a gel extraction kit.
(B-5) The DNA fragment obtained in (b-3) and the DNA fragment obtained in (b-4) were subjected to a ligation reaction (Ligation High, manufactured by Toyobo Co., Ltd.) at 16 ° C. to obtain Escherichia coli JM109 strain was transformed with the plasmid.
(B-6) The transformed solution was spread on an LB plate culture solution containing 20 μg / mL of kanamycin and 1% (w / v) of glucose, and a target clone was selected.
(B-7) A plurality of selected colonies were cultured and plasmid extraction was performed, and cleavage patterns with various restriction enzymes were confirmed.

  By the above method, A2820-FcR / pBBR1MCS2, which is a plasmid in which the H16_A2935 gene in the plasmid A2935-FcR / pBBR1MCS2 is replaced with the H16_A2820 gene, was obtained. A2820-FcR / pBBR1MCS2 is a plasmid in which a fazine promoter, a fusion gene of (H16_A2820 gene-cleavable linker gene-Fc binding protein gene) and a terminator gene are inserted in the positive direction with respect to LacZ of pBBR1MCS2.

(C) plasmid (c-1) H16_B0271 N-terminal region protein gene derived from Ralstonia eutropha H16 strain into which a fazine promoter, a fusion gene of (H16_B0271 N-terminal region gene-cleavable linker gene-Fc binding protein gene), and a terminator gene were introduced Plasmid B0271N / pUC57 obtained by inserting a polynucleotide (SEQ ID NO: 10) containing a nucleotide sequence (region 7 to 519 of SEQ ID NO: 10) obtained by conversion with a codon suitable for Ralstonia eutropha H16 strain into plasmid pUC57 Manufactured by artificial gene synthesis (Gen Script artificial gene synthesis contract service).
(C-2) Escherichia coli JM109 strain was transformed with the prepared B0271N / pUC57, and plasmid B0271N / pUC57 was extracted from the culture of the obtained transformant.
(C-3) A2935-FcR / pBBR1MCS2 prepared in (a) was digested with restriction enzymes HindIII and SpeI, and after agarose electrophoresis, the obtained DNA fragment of about 6.6 kbp was purified by a gel extraction kit.
(C-4) B0271N / pUC57 prepared in (c-2) was digested with restriction enzymes with HindIII and SpeI, and after agarose electrophoresis, the obtained DNA fragment of about 0.5 kbp was purified with a gel extraction kit.
(C-5) The DNA fragment obtained in (c-3) and the DNA fragment obtained in (c-4) were subjected to a ligation reaction (Ligation High, manufactured by Toyobo Co., Ltd.) at 16 ° C. to obtain Escherichia coli JM109 strain was transformed with the plasmid.
(C-6) The transformed solution was spread on an LB plate culture solution containing 20 μg / mL of kanamycin and 1% (w / v) of glucose, and the target clone was selected.
(C-7) A plurality of selected colonies were cultured and plasmid extraction was performed, and cleavage patterns with various restriction enzymes were confirmed.

  By the above method, B0271N-FcR / pBBR1MCS2, which is a plasmid in which the H16_A2935 gene in the plasmid A2935-FcR / pBBR1MCS2 was replaced with the H16_B0271N terminal region gene, was obtained. B0271N-FcR / pBBR1MCS2 is a plasmid in which a fazine promoter, a fusion gene of (H16_B0271N terminal region gene-cleaved linker gene-Fc binding protein gene) and a terminator gene are inserted in the positive direction with respect to LacZ of pBBR1MCS2. .

Example 2 Production of Ralstonia eutropha transformant A2820-FcR / pBBR1MCS2 produced in Example 1 (b), B0271N-FcR / pBBR1MCS2 produced in Example 1 (c), and Fc binding protein by the following method As a negative control not containing a gene, Ralstonia eutropha was transformed with a commercially available pBBR1MCS2 plasmid, respectively.
(1) Conjugative E. coli S17-1 was transformed with A2820-FcR / pBBR1MCS2, B0271N-FcR / pBBR1MCS2, or pBBR1MCS2.
(2) The transformed solution was spread on an LB plate culture solution containing 50 μg / mL of kanamycin, and a target clone was selected.
(3) The selected Escherichia coli S17-1 strain transformant was cultured at 37 ° C. in an LB (bactotryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) culture solution containing kanamycin 50 μg / mL. An overnight culture was performed.
(4) Commercially available Ralstonia eutropha H16 strain (ATCC 17699) or alstonia eutropha PHB_4 strain (DSM 541) in Nutrient Broth culture solution (Difco's Nutrient Broth, 8 g / L at 30 ° C.) Cultured overnight.
(5) The cells cultured in (4) are centrifuged and concentrated. After measuring the turbidity of the cells at 600 nm, the transformants of each E. coli S17-1 strain cultured in (3) and the cell turbidity 1 The mixture was mixed at a ratio of 1: 1 and plated on a nutrient broth plate (Nutrient Broth 8 g / L, bactogarose 15 g / L) containing no antibiotics, and cultured at 30 ° C overnight. As a control, transformants of each E. coli S17-1 strain, Ralstonia eutropha H16 strain or Ralstonia eutropha PHB_4 strain alone were cultured overnight at 30 ° C. on a nutrient broth plate containing no antibiotics.
(6) The bacterial cells on the surface of the plate are suspended in a nutrient broth culture solution, diluted to 1/1000 with a nutrient broth culture solution, and then applied to a nutrient broth plate containing 600 μg / mL kanamycin, starting at 30 ° C. for 2 days. Cultured for 3 days. Only 0 to several colonies appeared from a plate in which only the transformant of each Escherichia coli S17-1 strain, Ralstonia eutropha H16 strain or Ralstonia eutropha PHB — 4 strain, was cultured as a control. On the other hand, several tens to several hundreds of colonies were obtained from the plates coated with the mixed cells obtained by joining the transformant of Escherichia coli S17-1 and the strain of the genus Ralstonia.
(7) Each clone of the obtained conjugated cells was cultured and plasmid extraction was performed, and it was confirmed that the target plasmid was transferred by analysis of cleavage patterns with various restriction enzymes.

With the above method,
A2820-FcR / pBBR1MCS2 (plasmid prepared in Example 1 (b)) A2820-FcR / H16 strain, which is a strain obtained by conjugation to Ralstonia eutropha H16 strain,
A2820-FcR / PHB_4 strain, which is a strain obtained by conjugating A2820-FcR / pBBR1MCS2 (the plasmid prepared in Example 1 (b)) to the PHB_4 strain,
B0271N-FcR / H16 strain, which is a strain obtained by conjugating and transferring B0271N-FcR / pBBR1MCS2 (plasmid prepared in Example 1 (c)) to the H16 strain,
B0271N-FcR / PHB_4 strain, which is a strain obtained by conjugating B0271N-FcR / pBBR1MCS2 (plasmid prepared in Example 1 (c)) to Ralstonia eutropha PHB_4,
BBR1MCS2 / H16 strain, which is a strain obtained by conjugating pBBR1MCS2 (commercial plasmid) to Ralstonia eutropha H16 strain, and BBR1MCS2 / PHB_4 strain, which is a strain obtained by conjugating pBBR1MCS2 (commercial plasmid) to PHB_4 strain,
(Hereinafter, the strain is described with the above name).

Example 3 Evaluation of productivity of Fc binding protein in test tube culture Using the hydrogen-oxidizing bacteria prepared in Example 2, the secretion expression of Fc binding protein was evaluated.
(1) A2820-FcR / H16 strain, A2820-FcR / PHB_4 strain, B0271N-FcR / H16 strain, B0271N-FcR / PHB_4 strain, BBR1MCS2 / H16 strain, BBR1MCS2 / PHB_4 strain prepared in Example 2, respectively Pre-cultured in a test tube using 3 ml of 2 × YT (bactotryptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) containing 300 μg / mL kanamycin (30 ° C., 180 rpm, overnight) ).
(2) 150 μL of the preculture solution of (1) is inoculated into 3 ml of 2 × YT culture solution each containing 4% (w / v) sodium gluconate and 300 μg / mL of kanamycin, and 3 days at 30 ° C. and 180 rpm Culture was performed.
(3) 1 mL of the bacterial cells were collected in a microtube and centrifuged at 15000 rpm for 10 minutes to separate the supernatant and the bacterial cell pellet. The supernatant was aliquoted in 500 μL in separate microtubes, and 500 μL of 60% (v / v) glycerol aqueous solution was added and stored. On the other hand, the bacterial cell pellet from which the supernatant was removed was a protein extraction reagent containing 1 mL of protein extraction reagent (lysozyme 0.2 mg / mL, ethylenediaminetetraacetic acid 1 mM, phenylmethylsulfonyl fluoride 1 mM, Benzonase 125 U / mL). ) Was added to extract the protein in the cells.
(4) The amount of Fc binding protein in the supernatant and bacterial cells was measured by the ELISA method shown below.
(4-1) To a 96-well ELISA plate (manufactured by Nunc), add 10 μg / mL of gamma globulin preparation (manufactured by Chemo-Serum Therapy Laboratories) to each well and leave at 4 ° C. for 18 hours. Fixed.
(4-2) After washing with TBS buffer (0.2% (w / v) Tween 20, 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl), Tween 20 containing 1% BSA was removed. 200 μL each of TBS buffer (20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl) was added, and the mixture was allowed to stand at 4 ° C. for 18 hours or longer to perform a blocking operation.
(4-3) After washing with TBS buffer, 100 μL of the stored culture supernatant or dilution series of bacterial cell extract was added to each well and allowed to react with immobilized gamma globulin (30 ° C., 1 hour).
(4-4) After completion of the reaction, the well was washed with TBS buffer, and 100 μL of the primary antibody anti-hFcγR1 / CD64 antibody (MAB12571 manufactured by R & D) 0.1 μL / mL was added to each well for reaction (30 ° C. 1 hour).
(4-5) After completion of the reaction, the plate was washed with TBS buffer, and 100 μL of the secondary antibody Goat anti-Mouse IgG-h + I HRP antibody (ATH-216P manufactured by BETHYL) was added to each well for reaction (30 ° C. 1 hour).
(4-6) After completion of the reaction, the plate was washed with TBS buffer, and 50 μL of TMB Peroxidase Substrate (manufactured by KPL) was added to each well to cause a color reaction.
(4-7) After color development, 1M phosphoric acid aqueous solution was added to stop the reaction, and the absorbance at 450 nm was measured.

  The results are shown in FIG. None of the BBR1MCS2 / H16 and BBR1MCS2 / PHB_4 strains, which are negative controls (recombinant microorganisms into which no Fc-binding protein gene was introduced), produced Fc-binding protein. On the other hand, A2820-FcR / H16 strain, A2820-FcR / PHB_4 strain, B0271N-FcR / H16 strain, B0271N-FcR / PHB_4 strain, which are strains obtained by introducing an Fc binding protein gene and a transport protein gene into hydrogen-oxidizing bacteria, Fc-binding proteins were produced at 14 mg / L, 9 mg / L, 18 mg / L, and 7 mg / L, respectively, and the secretion ratios into the culture medium were 64%, 6%, 77%, and 27%, respectively. Thus, by changing the transport protein gene from H16_A2820 to H16_B0271, an improvement in the secretion ratio was observed when either the Ralstonia eutropha H16 strain or the PHB_4 strain was used as the host. In particular, in the culture of the B0271N-FcR / H16 strain, a result that exceeds both the productivity and secretion ratio of the A2820-FcR / H16 strain, which is the strain that showed the best productivity / secretion ratio in the previous reports, can be obtained. I was able to.

  Therefore, by introducing H16_B0271, which is a higher-performance transport protein gene, into the hydrogen-oxidizing bacterium together with the target protein gene, and co-expressing the target protein and the transport protein, the productivity of the target protein is improved, and It can be seen that the ratio of secretion into the culture medium is also improved.

Claims (5)

Transportation protein gene, the cleavage linker gene and a target protein gene, in this order a plasmid containing on the same plasmid is obtained by introducing the hydrogen-oxidizing bacteria, polypeptides wherein transporter protein consisting of the sequences set forth in SEQ ID NO: 8 A hydrogen-oxidizing bacterium Ralstonia eutropha H16 capable of expressing a protein. The hydrogen-oxidizing bacterium Ralstonia eutropha H16 according to claim 1, wherein the transport protein gene is a polynucleotide comprising the sequence set forth in SEQ ID NO: 10 . The hydrogen-oxidizing bacterium Ralstonia eutropha H16 according to claim 1 or 2 , wherein the target protein is an Fc-binding protein. A hydrogen-oxidizing bacterium Ralstonia eutropha H16 according to any one of claims 1 to 3 is cultured, and a gene containing a transport protein gene, a cleaved linker gene and a target protein gene in this order is expressed, and the transport protein , cleaved linker protein and target protein are expressed. To secrete and express the target protein outside the hydrogen-oxidizing bacterium Ralstonia eutropha H16 . Na Ru polynucleotide a sequence described in SEQ ID NO: 10, cutting linker gene and a target protein gene in this order comprises on the same plasmid, the hydrogen oxidizing bacteria Ralstonia eutropha H16 plasmid for secretory expression of the desired protein to the outside.

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